scholarly journals Facile Fabrication of Thin-Bottom Round-Well Plates Using the Deformation of PDMS Molds and Their Application for Single-Cell PCR

Micromachines ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 748
Author(s):  
Shinya Yamahira ◽  
Yuji Heike
Keyword(s):  
Single Cell ◽  
Low Cost ◽  
Science Research ◽  
Supporting Cell ◽  
Dimensional Error ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Single Cell Pcr ◽  
Novel Processing ◽  
Dimensional Errors

Recently, microdevices made of resins have been strongly supporting cell analysis in a range of fields, from fundamental life science research to medical applications. Many microdevices are fabricated by molding resin to a mold made precisely from rigid materials. However, because dimensional errors in the mold are also accurately printed to the products, the accuracy of the product is limited to less than the accuracy of the rigid mold. Therefore, we hypothesized that if dimensional errors could be self-corrected by elastic molds, microdevices could be facilely fabricated with precision beyond that of molds. In this paper, we report a novel processing strategy in which an elastic mold made of polymethylsiloxane (PDMS) deforms to compensate for the dimensional error on the products. By heat-press molding a polycarbonate plate using a mold that has 384 PDMS convexes with a large dimensional error of height of ± 15.6 µm in standard deviation, a 384-round-well plate with a bottom thickness 13.3 ± 2.3 µm (n = 384) was easily fabricated. Finally, single-cell observation and polymerase chain reactions (PCRs) demonstrated the application of the products made by elastic PDMS molds. Therefore, this processing method is a promising strategy for facile, low-cost, and higher precision microfabrication.

scholarly journals High-resolution within-sewer SARS-CoV-2 surveillance facilitates informed intervention

2021 ◽  
Author(s):  
Katelyn Reeves ◽  
Jennifer N. Liebig ◽  
Anontio Dimitri Feula ◽  
Tassa Saldi ◽  
Erika Lasda ◽  
...  
Keyword(s):  
Low Cost ◽  
Endogenous Control ◽  
Concentration Data ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
University Of Colorado ◽  
Key Stakeholders ◽  
Wastewater Samples ◽  
Critical Phases ◽  
The University

To assist in the COVID-19 public health guidance on a college campus, daily composite wastewater samples were withdrawn at 20 manhole locations across the University of Colorado Boulder campus. Low-cost autosamplers were fabricated in-house to enable an economical approach to this distributed study. These sample stations operated from August 25th until November 23rd during the fall 2020 semester, with 1,512 samples collected. The concentration of SARS-CoV-2 in each sample was quantified through two comparative reverse transcription quantitative polymerase chain reactions (RT-qPCRs). These methods were distinct in the utilization of technical replicates and normalization to an endogenous control. (1) Higher temporal resolution compensates for supply chain or other constraints that prevent technical or biological replicates. (2) The endogenous control normalized data agreed with the raw concentration data, minimizing the utility of normalization. The raw wastewater concentration values reflected SARS-CoV-2 prevalence on campus as detected by clinical services. Overall, combining the low-cost composite sampler with a method that quantifies the SARS-CoV-2 signal within six hours enabled actionable and time-responsive data delivered to key stakeholders. With daily reporting of the findings, wastewater surveillance assisted in decision making during critical phases of the pandemic on campus, from detecting individual cases within populations ranging from 109 to 2,048 individuals to monitoring the success of on-campus interventions.

scholarly journals Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits

2019 ◽  
Vol 9 (6) ◽  
pp. 4635-4641 ◽  
Keyword(s):  
Gold Nanoparticles ◽  
Nucleic Acids ◽  
Low Cost ◽  
Gc Content ◽  
Nucleic Acid Amplification ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Polymerase Chain ◽  
Successful Amplification

Various strategies have been suggested for successful amplification of the hard-to-amplify nucleic acids such as the inclusion of various chemical or biological materials in the in vitro nucleic acid amplification reactions, particularly polymerase chain reaction (PCR), and adjustment of the cycling programs. Although much efforts have been madefor improvements in the enhancement of polymerase chain reactions of high GC content nucleic acids, still significant challenges remain. In this study, the effects of citrate-coated AuNP and chamomile extract-coated AuNP (p-AuNP) on amplification of three genes with significant different GC percentage were evaluated. Owing to the enormous potential of gold nanoparticles in the enhancement of the PCR reactions, we showed the promising and consistent findings on the application of very dilute biocompatible chamomile-gold nanoparticles as safe and low-cost nanomaterials for molecular amplification of GC-rich DNA samples. We hypothesized that green AuNPs, which have different surface chemistry from cit-AuNPs, not only do not interfere with the PCR reactants but also are capable of enhancing PCR reactions. These results, for the first time, confirm the potential of using the green gold nanoparticles in the heat-assisted enzymatic in vitro reactions, suggesting Chamomile gold nanoparticles as reliable component of any PCR kits.

scholarly journals Investigation of Lethal Concurrent Outbreak of Chlamydiosis and Pigeon Circovirus in a Zoo

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1654
Author(s):  
Wei-Tao Chen ◽  
Chin-Ann Teng ◽  
Cheng-Hsin Shih ◽  
Wei-Hsiang Huang ◽  
Yi-Fan Jiang ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Chlamydia Psittaci ◽  
Bursa Of Fabricius ◽  
Chain Reactions ◽  
Renal Epithelium ◽  
Polymerase Chain Reactions ◽  
Transmission Electron ◽  
Intracytoplasmic Inclusion Bodies ◽  
Polymerase Chain ◽  
Streptopelia Orientalis

During the spring, an outbreak of sudden death involving 58 birds occurred in a zoo. Histopathological examinations revealed variable numbers of intracytoplasmic basophilic microorganisms in the macrophages, hepatocytes, and renal epithelium of most birds, along with occasional botryoid intracytoplasmic inclusion bodies within histiocytes in the bursa of Fabricius. Based on the results of histopathological examinations, immunohistochemical staining, transmission electron microscopy, and polymerase chain reactions, genotype B Chlamydia psittaci infection concurrent with pigeon circovirus (PiCV) was diagnosed. A retrospective survey, including two years before the outbreak and the outbreak year, of C. psittaci and PiCV infections of dead birds in the aviaries, revealed that the outbreak was an independent episode. The findings of this study indicate that concurrent infection with C. psittaci and PiCV might lead to lethal outbreaks of chlamydiosis, particularly Streptopelia orientalis. In addition, persistently monitoring both pathogens and identifying potential PiCV carriers or transmitters might also help prevent lethal disease outbreaks.

scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

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